Certain species of bacteria are unique in their ability to form metabolically dormant spores. Spore formation is usually the result of a response to nutrient exhaustion in the surrounding habitat of the cell and serves to protect the organism from harsh environments such as extreme heat, dehydration, UV and gamma-radiation and mechanical stress until conditions once again become suitable for metabolic activity to resume (Driks and Setlow, 2000). The most effective spore-formers are species of Bacillus and Clostridium which have been shown to remain dormant for millions of years as well as survive the harsh environments of outer space (Cano and Borucki, 1995; La Duc et al., 2004). Recently, certain spore-forming bacteria such as certain Bacillus species have been used as biowarfare agents (e.g., weaponized B. anthracis spores).
The morphology of a prokaryotic spore varies between species but for the most part consists of a condensed core, an inner spore membrane, a cortex, an inner and an outer spore coat and, in some cases, an exosporium (Santo and Doi, 1974). The spore coat is the outermost structure. It is common to all spores and is largely made up of protein (Kornberg et al., 1968). The composition of each layer is different and unique between spore types. For example, spores differ in the number and types of proteins in their coats, as well as the structure and modification of such proteins. The coat of B. cereus consists of one predominant coat protein present in a single layer, while the coat of B. subtilis contains upwards of 20 different proteins that contribute to two distinct coat layers (Aronson and Horn, 1976; Goldman and Tipper 1978, Pandey and Aronson, 1978; Jenkinson et al., 1981).
The spore cell wall is largely comprised of a thick cortex, consisting of two distinct types of peptidoglycan (Warth and Strominger, 1969, 1971, 1972). The germ cell wall is the layer closest to the inner forespore membrane. It has the same chemical composition as the mother cell and is believed to function as a template for peptidoglycan synthesis during spore germination. There is significant structural variation between species in the germ cell wall peptidoglycan. The structure of the cortex peptidoglycan, on the other hand, is similar in all species that have been examined to date, but it is slightly different from that of the mother cell. It has about 50% of its N-acetyl muramic acid residues substituted with muramic acid-delta-lactam, and has 10-12 fold less cross-linking between the glycan strands (Arith et al., 1996; Popham et al., 1996).
DNA exists within the spore core in a highly dehydrated state. Just prior to completion of spore maturation, the cell produces an over-abundance of small acid-soluble proteins (SASPs) (Nicholson and Setlow, 1990; Pogliano et al., 1995). The SASPs bind non-specifically to the DNA and, in dehydrated conditions, convert the DNA structure from its native B-form to an A-form (Griffith et al., 1994; Mohr et al., 1991; Setlow, 1992, Nicholson et al., 1990). It is this conformational change to DNA that has been shown to give the spore increased resistance to UV radiation (Fairhead and Setlow, 1992; Mohr et. al., 1991).
Isolation of DNA from prokaryotic spores would enable identification and analysis of such spores. Since spores are characteristically known for their resilience to chemical, enzymatic and mechanical damage, the methods previously used to lyse spores employed extreme and harsh measures that released DNA but also exposed it to harsh environments that compromised its structural integrity. The degree of damage to the isolated DNA was primarily dependent on the technique used to break open the spore and extract the genomic DNA. Because the spore is naturally highly resistant to enzymatic and chemical treatments, many methods have resorted to mechanical means for opening the spores and releasing the DNA. These techniques included boiling, French press, glass bead grinding, freeze/thaw cycles, and sonication (Taylor et al., 2001). Furthermore, mechanical disruption of spores to extract DNA was often preferred in order to avoid using chemicals and enzymes that would inhibit downstream PCR steps (Ivintski et al., 2003). These stresses however have a direct impact on the spore DNA once it is released and exposed to the same turbulent environment used to break open the spores. Even techniques used to purify DNA from the mixture of digested spore components (as well as from the large excesses of SASPs that coat DNA) can shear DNA. These steps include affinity chromatography columns, phenol/chloroform extraction, DNA precipitation by alcohols, vortexing and high speed centrifugation.
Accordingly, traditional methods of genomic DNA isolation from mature, non-germinating spores typically yielded fragments of DNA that averaged a few kilobases (kb) in length. DNA of this size is suitable for applications that rely on amplification of a specific section of DNA that is then used for DNA cloning, genetic analysis, etc. (Ivnitski, et. al., 2003). For manipulations involving polymerase chain reaction (PCR), the state and size of the harvested DNA was usually sufficient. However, for more detailed analysis on genome organization and/or phylogenic studies, the DNA isolated using these methods was insufficient.
There is a need for methods that rapidly and reliably yield high molecular weight nucleic acids from prokaryotic spores since such nucleic acids would facilitate analysis and identification of spores.